Ionizing radiation (IR) and cross-linking agents (CLA) induce DNA double-stranded breaks (DSB). DSB are the most harmful type of DNA damage, which causes genome instability leading to cancer, genetic diseases, and premature aging in human. However, IR and CLA, like cis-platin, are also commonly used in tumor therapy. Therefore, investigation of the mechanisms of DSB repair is crucially important. Recent discoveries have demonstrated that the cellular system of homologous recombination (HR) plays an essential role in DSB repair in all species. To insure the accuracy of DNA repair, the HR system uses homologous dsDNA as a template. Rad51 protein plays a key role in the search for homologous DNA sequences and for DNA pairing in all organisms. In eukaryotes, Rad51 protein carries out its function by forming transient complexes with other HR proteins that stimulate its DNA pairing activity. Among the stimulatory proteins, Rad54 protein is especially important. The long-term goal of this proposal is to contribute to understanding of the functions of the human HR protein complexes in DSB repair. The general approach is to characterize the specific functions of purified human HR proteins and investigate their interactions in vitro. Currently, we focus our investigation on the functions of human Rad54 protein in DNA repair. We will pursue the following specific aims. 1). We will study the biochemical properties of Rad54 protein and determine the mechanism of Rad54 protein binding to DNA. 2). We will investigate the interaction between Rad54 and Rad51, two most important proteins in DSB repair, and determine the mechanism how Rad54 protein enhances the ability of Rad51 protein to repair DSB. Reciprocally, we examine how interactions with Rad51 protein affect the properties of Rad54 protein. 3). We will examine the interaction of Rad54 protein with Rad51C protein, another HR protein, which has DNA pairing activity, albeit weak. 4). We will examine the biochemical activities of Rad54 protein mutants associated with human tumors. From all these studies the role of human Rad54 protein in DSB repair will be elucidated. The knowledge acquired in this study will help to better understand the fundamental mechanisms of HR and will be applied for developing efficient tumor therapies.